The primary goal of this project is to examine the role of reiterated DNA sequences in gene expression. Fifty percent of our efforts are directed towards examining the potential role of reiterated sequences in governing the expression of a family of related genes. For this purpose we intend to examine the possibility that specific classes of reiterated DNA sequences are associated with genes whose expression is modulated during development. A replica hybridization protocol using poly A plus mRNA from vegetative or developing cells will be used in the colony hybridization procedures to identify recombinant clones containing developmental genes. We will explore whether they have reiterated sequences by labeling them in vitro with 32P and annealing them to Southern blots of EcoR1 digested D. discoideum DNA. Clones containing reiterated DNA sequences will anneal to a complex pattern of bands. It will then be possible to use clones containing reiterative sequences as probes to locate "sister" clones. The common expression of these "sister" clones can then be examined in both vegetative and developing cells. If they share common temporal expression patterns it supports the notion that the repetitive sequences play a role in their expression. The balance of the program includes a detailed structural analysis of the reiterated ribosomal (rRNA) genes and several reiterated transfer RNA (tRNA) genes. We expect to complete the sequence analysis in D. discoideum. The initial results support the notion that D. discoideum as measured by rRNA primary structure has diverged over tremendous evolutionary distances from both Yeast and mammalian cells. The analysis of tRNA genes has demonstrated the existence or association of reiterated DNA sequences with a Valine and a Tryptophan gene. In the next twelve months we expect to establish if reiterated DNA sequences always flank tRNA genes in D. discoideum.